Cell isolation and screening device and method of using same

ABSTRACT

The present invention provides methods and devices for screening a single cell or a small group of cells for a desired biological activity. In particular, the present invention provides for delivering cell(s) to a plurality of cell isolation regions of a cell isolation device, transferring cell(s) to a plurality of wells of a cell expansion device and then detecting the potential desired biological activity of the cell(s). Each of the receptacles comprise a recess sized to isolate a single cell or small group of cells and each of the wells encompass a cavity that provides sufficient volume for cell proliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 10/084,063, filed Feb. 28, 2002 and claims priorityto Provisional U.S. Application No. 60/334,593 filed Dec. 3, 2001, andProvisional U.S. Application No. 60/307,843 filed Jul. 27, 2001, [andU.S. application Ser. No. 10/084,063 filed Feb. 28, 2002] all of whichare herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to devices and methods that are useful forisolating and screening cells for a desired biological activity. Inparticular, the present invention relates to devices and methods thatare useful in isolating and screening hybridoma cells for specificantibody production.

BACKGROUND OF THE INVENTION

One of the major challenges for performing cell based screening is theisolation of small populations of cells in a manner that allows forsubsequent screening procedures. Traditional devices and methods ofisolating cells do not adequately provide for the isolation of smallpopulations of cells without performing steps that potentially modifycellular function or activity. Isolation of cells is not only importantin screening, but also in processes that involve the monitoring,measuring, and/or use of the output of cellular activity or function(e.g. antibody production) for small populations of cells.

For example, with respect to antibody production, one of the approachesused to produce antibodies is to manufacture hybridomas. These are cellscreated by the fusion of antibody-secreting B-cells and myeloma cells.This method is variable with respect to almost all stages of the processincluding the duration of screening and required needs for cell growthand proliferation. Much of the variability can be attributed to theimmunogen and immune response mounted by the immunized animal. Moreoverthis process is time consuming and labor intensive.

In general, once the fusion is performed and the cells are plated, thereare several issues that have to be addressed. First, the cells will growat different rates, thus the point at which one must perform the assayfor antibody production to assess positive pools of cells can vary andmay require more than one assay point on the same pool of cells. Duringthis process, the rapidly growing cells need to be passaged in order topromote viability and to prevent loss of potentially positive clones.The next step is to perform limiting dilution with the goal of achievingclonal populations. Successive rounds of this process may be required toachieve clonal or near clonal populations.

The value of specific monoclonal antibodies as useful tools fordiagnostics and immuno-therapies has meant that investigators have hadno alternative but to tolerate long production periods and highdevelopment costs. Rapid and efficient ways of screening hybridoma celllines for antibody production are not presently available. Therefore,there is a need for methods and devices to accelerate monoclonalantibody production for experimental, diagnostic and therapeuticapplications. The devices and methods disclosed herein dramaticallyshorten the period for monoclonal antibody production from a period ofweeks to a period of days. The invention provides a multicomponentdevice that also has application for other cell types requiring theisolation of a small population of cells and requiring the subsequentclonal expansion of this population of cells.

SUMMARY OF THE INVENTION

The present invention relates to devices and methods for screening acell or small group of cells for a desired biological activitycomprising a cell delivery step, a cell isolation device and acorresponding cell isolation step, a cell expansion device and acorresponding cell expansion step, and a detection device and acorresponding detection step.

In particular, the present invention provides a method of screeningcells for a desired biological activity comprising providing a cellisolation device defining a plurality of cell isolation regions, eachcell isolation region encompassing a recess, each of the cell isolationregions being sized to isolate about one to about five cells therein,and the cell isolation regions further defining a predetermined pitchwith respect to one another. The method further comprises delivering theabout one cell to about five cells to each of the cell isolationregions. The method additionally comprises providing a cell expansiondevice defining a plurality of wells corresponding to respective ones ofthe plurality of cell isolation regions, the wells defining apredetermined pitch matching the predetermined pitch of the cellisolation regions. The method also comprises transferring the about onecell to about five cells from the cell isolation regions to the wellsand allowing the about one cell to about five cells to proliferate andexhibit a desired biological activity in the wells. The method furthercomprises assaying the desired biological activity of the one cell toabout five cells.

The present invention also provides a kit for screening cells for adesired biological activity, the kit comprising a cell isolation devicedefining a plurality of cell isolation regions, each cell isolationregion encompassing a recess, each of the cell isolation regions beingsized to isolate about one to about five cells therein, and the cellisolation regions further defining a predetermined pitch with respect toone another. The kit further comprises a cell expansion device defininga plurality of wells corresponding to respective ones of the pluralityof cell isolation regions, and the wells defining a predetermined pitchmatching the predetermined pitch of the cell isolation regions.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given hereinbelow and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein:

FIG. 1 is a top perspective view of an embodiment of a cell deliverydevice and an embodiment of a cell isolation device according to thepresent invention.

FIG. 2 is a perspective view of an embodiment of a cell delivery deviceaccording to the present invention in contact with an embodiment of acell isolation device according to the present invention.

FIG. 3 is a schematic view of the channel pathways of an embodiment ofthe cell delivery device according to the present invention.

FIG. 4 is an oblique perspective view of an embodiment of a cellisolation device according to the present invention.

FIG. 5 is a cross-sectional view of the device of FIG. 4 along linesII—II.

FIG. 6 is a cross-sectional view of the device of FIG. 4.

FIG. 7 is an oblique perspective view of an alternative embodiment of acell isolation device according to the present invention.

FIG. 8 is a partially cut-away perspective view of an embodiment of acell isolation device according to the present invention.

FIG. 9A is a cross-sectional view of the cell isolation device of FIG. 8along lines III—III showing an embodiment of the cell isolation regionsof the cell isolation device.

FIG. 9B is a view similar to FIG. 8 showing an alternative embodiment ofthe cell isolation regions of the cell isolation device.

FIGS. 10A–E shows respective stages of a method of fabricating anembodiment of the cell isolation device according to the presentinvention.

FIG. 11 depicts a method of fabricating an alternative embodiment of acell isolation device according to the present invention.

FIGS. 12A–C show respective stages of a method of fabricating anembodiment of a cell isolation device according to the presentinvention.

FIGS. 13A–C shows respective stages of a method of fabricating anembodiment of a cell isolation device according to the presentinvention.

FIG. 14 is a cross-sectional view of an embodiment of a cell expansiondevice according to the present invention.

FIG. 15A shows a method of transferring cells from the cell isolationdevice to an embodiment of the cell expansion device according to thepresent invention.

FIG. 15B shows a method of transferring cells from the cell isolationdevice to an alternative embodiment of the cell expansion deviceaccording to the present invention.

FIG. 16 depicts a cross-sectional view of an embodiment of the detectiondevice and a method of detecting antibody production according to thepresent invention.

FIG. 17 is a cross-section view of an alternative embodiment of a cellexpansion device according to the present invention.

FIG. 18 shows a method of detecting antibody production according to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and devices for isolating asmall population of cells and for screening a larger population ofproliferated cells for biological activity with a minimal amount ofmanipulation of the cells. Although the present invention contemplatesthe screening of all types of cells for the detection of any desiredbiological activity, for purposes of illustration, the methods anddevices described herein are occasionally described in the context ofisolating and screening hybridoma cells for specific antibodyproduction. The present invention is not in any way limited to thisapplication and may be used to isolate any types of cells, including,but not limited to, cell lines that express or produce proteins,carbohydrates, enzymes, peptides, hormones, receptors; other cell linesthat produce antibodies; genetically engineered cells; and activatedcells. Moreover, the present invention may be used to screen for avariety of biological activities including, but not limited to, theexpression of surface receptor proteins, enzyme production, and peptideproduction. Furthermore, the present invention may be used to screen avariety of test agents to determine the effect of the test agents on thedesired biological activity. Other types of cells desired to be isolatedand screened, other types of biological activity desired to be detected,and specific test agents to be screened will be readily appreciated byone of skill in the art.

In general, the present invention relates to a method and accompanyingdevice for isolating and screening cells including a cell delivery step,a cell isolation step utilizing a cell isolation device, a cellexpansion step utilizing a cell expansion device, and a detection steputilizing a detection device. In general, cells are delivered to cellisolation regions of the cell isolation device. By “cell isolationregion” is meant any area capable of attracting, trapping, securingand/or binding a single cell or a small population of cells. Cell arethen transferred to wells of a cell expansion device. By “well” is meanta cavity that is capable of retaining cells therein. Cells are thentransferred from the cell expansion device to the detection device wherebiological activity is detected, or, in the alternative, the detectiondevice is brought into contact with the cell expansion device.

With respect to particular details of the methods and devices accordingto the present invention, in the cell delivery step, cells may bedelivered to the cell isolation regions of the cell isolation device(described in more detail below) by any means known in the art. Forexample, cells may be delivered by directly pipetting cells into or onthe cell isolation regions of the cell isolation device either manuallyor by robotic liquid handling systems; bulk delivering the cells influid to the cell isolation device and removing the excess by pipettingso as to leave only cells in or on the cell isolation regions; or byutilizing a delivery device with microfluidic channels to deliver thecells into or on the cell isolation regions.

FIG. 1 illustrates an example of a microfluidic delivery device 200 withmicrofluidic channels 220 to deliver the cells into or on cell isolationregions 20 of cell isolation device 10. “Microfluidic delivery device,”as used herein, refers to a system or device having fluidic conduits,such as channels, that are typically fabricated at the micron tosubmicron scale. Generally, the fluidic conduits have at least onecross-sectional dimension in the range of from about 0.1 μm to about 500μm. As seen in FIG. 1, the microfluidic device 200 generally comprises ahousing 210 defining a plurality of channels 220. Each channel includesan entrance port 230 at one end and an exit port 240 at another end.Although FIG. 1 depicts only two channels, microfluidic device 200 caninclude more than two channels 220 as seen in FIG. 3 (wherein dashedlines indicate channel paths and arrows indicate potential flow inmultiple directions through each channel). The diameter of channels 220should be large enough to prevent clogging of channels 220, preferablybeing about three times the diameter of the cell. A 100 μm×100 μmchannel is the preferred size yet alternate sizes may include, but arenot limited to, 60 μm×100 μm or 30 μm×100 μm. Although a microfluidicdevice for use in the present invention is described briefly below,methods of employing microfluidics for cell delivery are well-known inthe art and are described, for example, in Love, et al., MRS BULLETIN,pp. 523–527 (July 2001) “Fabrication of Three-Dimensional MicrofluidicSystems by Soft Lithography,” Delamarche et al,: JOURNAL OF AMERICANCHEMICAL SOCIETY, Vol. 120, pp. 500–508 (1998), Delamarche et al,:SCIENCE, Vol. 276, pp. 779–781 (May 1997), Quake et al., SCIENCE, Vol.290, pp. 1536–1540 (Nov. 24, 2000), all of which are hereby incorporatedby reference.

With respect to the use of microfluidic device 200 according to thepresent invention, as seen in FIG. 2, microfluidic device 200 iscontacted with the top surface of cell isolation device 10 to provide asubstantially fluid-tight seal. Cell are delivered to the cell isolationregions by flowing medium containing the cells desired to be isolatedthrough channels 220 over cell isolation regions 20. The flowing of themedium containing the cells through channels 220 of microfluidic device200 may be carried out by a number of mechanisms, including, forexample, pressure based flow, electrokinetic flow, or mechanisms thatutilize a hybrid of the two. Microfluidic device 200 may also includeintegrated microfluidic structures, such as micropumps and microvalves,or external elements, e.g., pumps and switching valves, for the pumpingand direction of the medium through device 200. Medium transport anddirection may also be accomplished through electroosmosis orelectrokinesis. The selection and details of the above mechanisms wouldbe well within the knowledge of a person skilled in the art.

In some embodiments of the present invention, preferably the flow of thecell containing medium into the cell isolation regions is constant, orcontinuous. Typically, a known and/or constant flow rate is useful, forexample, when attempting to establish precise control over the deliveryof cells to cell isolation regions 20. Furthermore, constant flowfacilitates feeding of cells and high throughput screening.

In one embodiment of the method of delivering cells to cell isolationdevice 10 according to the present invention, microfluidic device 200 isused to deliver about fifty to about hundred cells to each cellisolation region 20. In another embodiment, microfluidic device 200 isused to deliver about twenty-five to about fifty cells to each cellisolation region 20. In yet another embodiment, microfluidic device 200is used to delivery about ten to about twenty-five cells to each cellisolation region 20. In another embodiment, microfluidic device 200 isused to delivery about five to about ten cells to each cell isolationregion 20. In still another embodiment, microfluidic device is used todelivery about one to about five cells to each cell isolation region 20.In a preferred embodiment, microfluidic device is used to deliver asingle cell to each cell isolation region 20. Typically, about 90% ofeach of the cell isolation regions 20 of the cell isolation device 10are filled when the flow rate is adjusted or regulated. The flow rate bywhich the cell containing medium is delivered to the cell isolationregions 20 can be adjusted by known methods. A person of skill in theart would know how to adjust the flow rate of a microfluidic device toachieve a desired cell distribution per cell isolation region 20.

Microfluidic device 200 may be fabricated from materials that arecompatible with the conditions present in the particular experiment ofinterest. Such conditions include, but are not limited to, pH,temperature, ionic concentration, pressure, and application ofelectrical fields. The materials of device 200 may also be chosen fortheir inertness to components of the experiment to be carried out in thedevice. Such materials include, but are not limited to, glass, silicon,fused silica, metal films, polymeric substrates, such as polystyrene,poly(methylacrylate), polydimethylsiloxane (PDMS) and polycarbonate,depending on the intended application.

Notwithstanding the method of delivery, according to embodiments of thepresent invention, cells are ultimately delivered to cell isolationdevice 10 where cells are isolated. As illustrated in FIG. 4 and FIG. 7,cell isolation device 10(a/b) utilized in the cell isolation stepgenerally includes a plurality of cell isolation regions 20(a/b) forreceiving cells therein.

FIGS. 4–6 show one embodiment of a cell isolation device according tothe present invention, where the cell isolation device 10 a generallyincludes a housing 11 defining a plurality of cell isolation regions 20a into which cells 30 may be delivered. Cell isolation region 20 apresents a recess sized to isolate a single cell 30 or a small group ofcells 30. In one embodiment, cell isolation region 20 a is sized toisolate about fifty to about hundred cells. In another embodiment, cellisolation region 20 a is sized to isolate about twenty-five to aboutfifty cells. In yet another embodiment, cell isolation region 20 a issized to isolate about ten to about twenty-five cells. In anotherembodiment, cell isolation region 20 a is sized to isolate about five toabout ten cells. In still another embodiment, cell isolation region 20 ais sized to isolate about one to about five cells. In a preferredembodiment, cell isolation region 20 a is sized to isolate a singlecell.

In another embodiment of the cell isolation device, as seen in FIG. 7,cell isolation device 10 b is generally defined by a surface 40 defininga plurality of cell isolation regions 20 b upon which cells 30 may bedelivered. Preferably, surface 40 is planar. In the embodiment depictedin FIG. 7, cell isolation region 20 b, corresponds to a discrete area ofsurface 40 that has the ability to attract, trap, secure and/or bind acell 30 or a small group of cells 30 thereon with respect to thenon-attractive surrounding surface 40 (such discrete area referred toherein as a “bioaffinity region”). This ability to secure cells 30 tothe bioaffinity region may be accomplished through the use of“bioaffinity ligands” as disclosed in more detail herein. With respectto the size of cell isolation region 20 b, in one embodiment, cellisolation region 20 b is sized to isolate about fifty to about hundredcells. In another embodiment, cell isolation region 20 b is sized toisolate about twenty-five to about fifty cells. In yet anotherembodiment, cell isolation region 20 b is sized to isolate about ten toabout twenty-five cells. In another embodiment, cell isolation region 20b is sized to isolate about five to about ten cells. In still anotherembodiment, cell isolation region 20 b is sized to isolate about one toabout five cells. In a preferred embodiment, cell isolation region 20 bis sized to isolate a single cell.

Notwithstanding the type of cell isolation region 20 utilized,preferably adjacent cell isolation regions 20 are disposed relative toone another to define a predetermine pitch. More, preferably, the cellisolation regions 20 are disposed relative to one another to match apitch P of an industry standard microtiter plate such as, for example, a24-, 96-, 384-, 768-, or a 1536-well microtiter plate. The term “pitch”P as used herein in reference to the cell isolation device, the cellexpansion device, and the cell detection device, refers to the distancebetween respective vertical centerlines between adjacent cell isolationregions 20 in the test orientation of the particular device beingutilized. By “test orientation” of the device is meant to refer to aspatial orientation of the cell isolation device of the particulardevice being utilized during its respective step. For example, the testorientation of the cell isolation device 10 is the orientation of thedevice when cells are isolated in their respective cell isolationregions 20. The test orientation of the cell expansion device is theorientation of the cell expansion device when cells are allowed toproliferate in their respective wells. It is also noted that in thecontext of the present invention, “top,” “bottom,” and “lateral” aredefined relative to the test orientation of the cell isolation device.

Although the present invention contemplates both the isolation of asingle cell 30 and small groups of cells 30, in certain embodiments itmay be preferable to isolate a single cell 30. Examples of suchembodiments include the embodiments of FIGS. 9A and 9B, whichrespectively show alternative embodiments of cell isolation region 20 a.Referring to FIGS. 9A and 9B, the shown embodiments of cell isolationdevice 10 a, show cell isolation region 20 a that is defined by amicrowell portion 52. The microwell portion 52 may adjoin accessorydelivery portions including a conical portion 51 Additionally, in theembodiments of FIGS. 8 and 9A, another accessory delivery portion, topcylindrical portion 50, may adjoin conical portion 51, which in turn,adjoins the bottom microwell portion 52. The dimensions of the accessorydelivery portions 51 and 52 and the cell isolation region, microwellportion 52, may include any dimensions that allow for cells to be addedto the cell isolation regions 20 a en masse yet permit only a singlecell to settle into microwell portion 52 of cell isolation region 20 a.Preferably, as depicted in FIG. 9B, the cylindrical portion 50 (inembodiments including portion 50) has a diameter d₁ of about 2millimeters (mm) and a depth D₁ of about 3 mm; the conical portion 51has a depth D₂ of about 1 mm; and the microwell portion 52 has adiameter d₃ of about 10 microns (μm) to about 50 μm and a depth D₃ ofabout 10 μm to about 50 μm. In a preferred embodiment, the diameter d₃of microwell portion 52 is about 20 μm and the depth D₃ of microwellportion is about 20 μm. The cell isolation regions 20 a may also bedisposed relative to one another to match a pitch of a standardmicrotiter plate such as, for example, a 24-, 96-, 384-, 768-, or a1536-well microtiter plate.

The present invention contemplates several methods of fabricating cellisolation device 10 a wherein cell isolation region 20 a of device 10 ais defined a microwell portion 52 and accessory delivery portions suchas conical portion 51 and optionally cylindrical portion 50. Referringto FIGS. 10(A–E), various stages of an embodiment of a fabricationmethod according to the present invention are shown. According to theshown embodiment of the fabrication method, device 10 a may befabricated by standard photolithographic procedures. According to thisprocedure, a substrate 70, as seen in FIG. 10A, defining a plurality ofconical elements 71 thereon extending from a base 72 is fabricated viastandard machining methods. The dimensions of each conical element 71corresponds to the dimensions of a corresponding conical portion 51 andmay be machined directly into base 72 or attached to base 72 bymechanical means, such as by press-fitting, or chemical means, such asby adhesive bonds. Alternatively, conical elements 71 may be fabricatedby a multi-level photolithography technique resulting in a quasi-conicaltopology of sequentially decreasing layers of photoresist as seen inFIG. 10E. The final layer 77 of photoresist corresponds to thedimensions of microwell portion 52. In a preferred embodiment and asillustrated in FIGS. 10A–E and 11, conical elements 71 are disposedrelative to one another to match a pitch of a standard microtiter plate.In an alternative embodiment (not shown), substrate 70 is furtherdefined by a plurality of cylindrical elements. With respect to a singlyrepresentative cylindrical element, the cylindrical element ispositioned between base 72 and a conical element 71. The dimensions ofthe cylindrical elements correspond to the dimensions of cylindricalportions 50. In such an embodiment, the substrate 70 may be used as thesubstrate in subsequent stages of FIGS. 10A–D described below.

As illustrated in FIG. 10B, in a further step of this fabricationprocedure, substrate 70 is coated with a photoresist 73 and asillustrated in FIG. 10C, a mask 74 defining regions 75, the dimensionsof which correspond to the dimensions of microwell portion 52, is placedover photoresist 73. Ultraviolet (UV) light is then exposed through mask74 onto photoresist 73 and the exposed photoresist is washed with adeveloper solution resulting in the formation of conical-microwellmaster 76 as seen in FIG. 10D. Material for the cell isolation device 10a is then either spin cast, injected, or molded over conical-microwellmaster 76 and cured, resulting in formation of the conical-microwellmember 80 as seen in FIG. 11. Photolithographic techniques arewell-known to one of skill in the art and as such, substrate 70materials, photoresist 73 materials, mask 74 materials, developersolutions, the use thereof, including variations of described technique,are well within the knowledge of one of skill in the art.

Referring to FIG. 11, in embodiments where it is desired to only haveeach cell isolation region 20 a defined by microwell portion 52 and toonly have conical portion 51 as the accessory cell delivery portion,such as the embodiment of FIG. 8B, then conical-microwell member 80comprises the entire single cell isolation device 10 a. In thealternative, in embodiments where it is desired to have accessorydelivery portions, cylindrical portion 50 and conical portion 51, suchas the embodiment of FIG. 8A, then a member 81 defining a plurality ofcylindrical through-holes 82 may be utilized as illustrated in FIG. 11.In this embodiment, member 81 is aligned with member 80 such that whenmember 81 is contacted with member 80, cylindrical through-holes 82 arebrought into registration with conical elements 71 to form the pluralityof accessory delivery portions (cylindrical portion 50 and conicalportion 51) adjoining cell isolation region (microwell portion 52).

As shown in FIGS. 12A–12C, another embodiment of a method of fabricatinga cell isolation device 10 a having cell isolation region 20 a adjoiningaccessory delivery portions of is through the use of surface tensiondriven epoxy molding. According to this embodiment, a substrate 70similar to the one described in relation to FIGS. 10A–E may be used.Here, conical elements 71 of substrate 70 are immersed into a liquid 90such as, for example, UV-curable epoxy as shown in FIG. 12A. Liquiddrops 72 form at the tips of conical elements 71 and these liquid drops72 are contacted with a support 91 as shown in FIG. 12B. Surface tensionforces cause the liquid drops 72 to take on a known geometry, such as acylindrical geometry. It will be recognized by one of skill in the artthat the conical elements 71 should be properly aligned and oriented inrelation to support 91 to assure uniformity of the liquid cylindricalshape. The liquid 72 is then cured into solid form resulting in theformation of a conical-microwell master 92 as seen in FIG. 12C. Materialfor the cell isolation device 10 a is then either spin cast, injected,or molded over cell isolation region master 92 resulting in theformation a conical-microwell member 80 as shown by way of example inFIG. 11, and as further shown in the embodiment of FIG. 9B.

As shown in FIGS. 13A–13C, an alternative method of fabricating a cellisolation device 10 a according to the present invention, is through theuse of epoxy molding in microfabricated wells. This method involves theuse of a substrate 70 similar to the one described in relation to FIGS.12A–C and a microwell housing 100 as seen in FIG. 13A. Microwell housing100 may be fabricated of photoresist, or cast in an elastomer such asPIMS. Microwell housing 100 defines a plurality of microwell 101therein, each of which has dimensions that correspond to the dimensionsof microwell portion 52. Preferably, the microwell 101 are disposedrelative to one another to match a pitch of a standard microtiter plate.Microwell 101 are filled with liquid 102, such as, for example,UV-curable epoxy. If needed, excess liquid can be removed from the areasof microwell housing 100 surrounding microwell 101 by any means known inthe art such as, for example, using a “squeegee” or “doctor blade,” orrazor thereby leaving only liquid in microwell 101. As shown in FIG.13B, conical elements 71 of substrate 70 and microwell 101 of microwellmember 100 are aligned such that conical elements 71 are in registrationwith their corresponding microwell 101. Conical elements 71 are thenimmersed into their respective microwell 101 and the liquid insidemicrowell 101 is cured into a solid form. Conical elements 71 are thenremoved from their respective microwell 101 resulting in the formationof conical-microwell master 92 as seen in FIG. 12C. Material for thecell isolation device 10 a is then either spin cast, injected, or moldedover cell isolation region master 92 to form conical-microwell member80.

Materials for cell isolations device 10 a include any rigid or flexiblemachinable material such as glass, co-polymer or polymer, mostpreferably urethane, rubber, molded plastic, polymethyl methacrylate(PMMU), polycarbonate, polytetrafluoroethylene (TEFLON), polyvinylchloride (PVC), polydimethylsiloxane (PIMS), polysulfone, and the like.

Cell isolation device 10 a (whether designed to isolate a single cell ora group of cells) may be exposed to differential surface treatment inorder to ensure that cells are isolated in cell isolation region 20 a.For example, the areas of housing 11 surrounding cell isolation regions20 a may be treated to reduce protein adhesion while cell isolationregions 20 a may be treated to provide a more hydrophilic environment.The hydrophilic nature of cell isolation region 20 a versus thesurrounding hydrophobic housing 11 would favor the beading of liquidcontaining cells 30 in cell isolation region 20 a and potentially holdcells 30 in place, limiting the exhibition of biological activity, suchas antibody deposition, to the “wet” areas. Examples of some hydrophobicsurface treatments known in the art are teflon, perfluoronated plastic,and polyethylene glycol.

In another embodiment of the cell isolation device 10 according to thepresent invention, cell isolation device 10 b comprises a surface 40having a plurality of cell isolation regions 20 b as seen in FIG. 7 forreceiving cells 30 thereon. In particular, cell isolation region 20 bcorresponds to a bioaffinity region upon which cells are immobilized.The ability to secure cells 30 to the bioaffinity region according tothe embodiment of FIG. 7 may be accomplished through the use ofbioaffinity ligands that are immobilized on the surface of thebioaffinity region and that are capable of binding to cells 30. The term“bioaffinity ligand” is defined herein to mean any biological or otherorganic molecule capable of specific or nonspecific binding orinteraction with another biological molecule (including biologicalmolecules on the surface of cells). Such binding or interaction may bereferred to as “ligand/ligate” binding or interaction and is exemplifiedby, but not limited to, antibody/antigen, antibody/hapten,enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, bindingprotein/substrate, carrier protein/substrate, lectin/carbohydrate,receptor/hormone, nucleic acid/nucleic acid, oligonucleotides/nucleicacid, receptor/effector or repressor/inducer bindings or interactions.Examples of bioaffinity ligands used herein, include but are not limitedto antibodies, Self-Assembled Monolayers (“SAMs”) with biospecificligands, lectins (e.g. Ulex europaeus I lectin which binds to theterminal L-fucosyl residues present on the surface of human endothelialcell; peanut agglutinin, agglutinin-I, and phytohaem agglutinin),carbohydrates, antigens, and lipid bilayers. The bioaffinity region ofcell isolation region 20 b may be coated with bioaffinity ligands toeither specifically or non-specifically bind the plasma membrane ofcells 30.

With respect to non-specific affinity, exemplary bioaffinity ligandshaving a general non-specific affinity to cells are the lanthanides andin particular, erbium, which is known to bind cell surface glycoproteinsas well as calcium-receptors. Another bioaffinity ligand having ageneral non-specific affinity to cells is ferritin.

In another embodiment, the bioaffinity ligand is an antibody specificfor mammalian cells. For example, anti-Ig kappa light chain antibody,anti-CD45R antibody, or anti-syndecan, may be used to differentiallybind activated B-cells. Preferably, an antibody specific for anti-Igkappa light chain antibody cells is used. Any of the methods known inthe art for conjugating an antibody to a solid phase support, such assurface 40 described herein, can be used in the present invention.

The use of SAMs provides a preferred method for binding and isolatingcells 30 in the bioaffinity region of cell isolation region 20 b. SAMsare the most widely studied and best developed examples ofnonbiological, self-assembling systems. They form spontaneously bychemisorption and self-organization of functionalized, long-chainorganic molecules onto the surfaces of appropriate substrates. SAMS areusually prepared by immersing a substrate in the solution containing aligand that is reactive toward the surface, or by exposing the substrateto the vapor of the reactive species. As is well-known to one of skillin the art, there are many systems known in the art to produce SAMs. Incertain embodiments, it may be desirable to pattern the SAMs to have anarrayed surface. Patterning SAMs on a planar surface has been achievedby a wide variety of techniques, including micro-contact printing,photo-oxidation, photo-cross-linking, photo-activation,photolithography/plating, electron beam writing, focused ion beamwriting, neutral metastable atom writing, SPM lithography,micro-machining, micro-pen writing. A preferred method is micro-contactprinting is described in U.S. Pat. No. 5,776,748 and is hereinincorporated by reference in its entirety.

With respect to the dimensions of cell isolation region 20 b accordingto the embodiment of FIG. 7, cell isolation regions 20 b of celldelivery device 10 b may be between 10 μm to 30 μm in diameter andpreferably between about 10 μm to about 15 μm in diameter and may bedisposed relative to one another to match a pitch of a standardmicrotiter plate.

As with cell isolation device 10 a, cell isolation device 10 b may beexposed to differential surface treatment in order to ensure that cellsare isolated in cell isolation region 20 b. For example, the areas ofsurface 40 surrounding cell isolation regions 20 b may be treated, forexample, with a hydrophobic surface treatment, to reduce proteinadhesion. The bioaffinity region of cell isolation region 20 a and thesurrounding hydrophobic area of surface 40 would favor the binding ofcells 30 on cell isolation region 20 b. Examples of some hydrophobicsurface treatments known in the art are teflon, perfluoronated plastic,and polyethylene glycol.

Although the description of the foregoing embodiments has focused on theuse of bioaffinity ligands to bind cells 30 onto cell isolation region20 b of cell isolation device 10 b, it is also contemplated by thepresent invention that bioaffinity ligands may be immobilized on thebottom surface of cell isolation regions 20 a of cell isolation device10 a to potentially bind cells 30.

With respect to surface 40 of cell delivery device 10 b, surface 40 maybe fabricated of any material capable of having bioaffinity ligandsimmobilized therein and such materials are readily known to one of skillin the art. Preferably, surface 40 is fabricated of any rigid orflexible machinable material such as glass, co-polymer or polymer, mostpreferably urethanes, rubber, molded plastic, polymethylmethacrylate(PMMA), polycarbonate, polytetrafluoroethylene (TEFLONTM),polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, andthe like. Such substrates are readily manufactured from microfabricatedmasters, using well known molding techniques, such as injection molding,embossing or stamping, or by polymerizing the polymeric precursormaterial within a mold. Standard soft lithography techniques may also beused to fabricate surface 40 (see e.g., Love, et al., MRS BULLETIN, pp.523–527 (July 2001) “Fabrication of Three-Dimensional MicrofluidicSystems by Soft Lithography,” Delamarche et al,: JOURNAL OF AMERICANCHEMICAL SOCIETY, Vol. 120, pp. 500–508 (1998), Delamarche et al,:SCIENCE, Vol. 276, pp. 779–781 (May 1997), Quake et al., SCIENCE, Vol.290, pp. 1536–1540 (Nov. 24, 2000), U.S. Pat. No. 6,090,251, all ofwhich are hereby incorporated by reference). Such surface materials arepreferred for their ease of manufacture, low cost and disposability, aswell as their general inertness to most extreme reaction conditions.These materials may include treated surfaces, such as, derivatized orcoated surfaces, to enhance their utility in the fluidic, preferablymicrofluidic, system, to provide enhanced fluid direction (See e.g.,U.S. Pat. No. 6,238,538, and which is incorporated herein by reference).

Once cells 30 are isolated in cell isolation regions 20 of cellisolation device 10, another aspect of the invention allows for theproliferation of the cells 30 through the use of a cell expansiondevice. As illustrated in FIG. 14, the cell expansion device 120comprises a structure 121 defining a plurality of wells 122 withadjacent wells having the same pitch as the pitch defined by adjacentcell isolation regions of the cell isolation device 10. In particular,each well 122 in a test orientation of the cell expansion device 120,encompasses a cavity surrounded by two lateral sides connected to abottom side. Preferably, each well is also characterized by having agreater volume than respective ones of the cell isolation region 20.“Wells” as used herein are specific to the cell expansion device,whereas cell isolation regions as used herein are specific to the cellisolation device. In general as seen in FIG. 14, cell expansion device120 is utilized by orienting structure 121 with housing 11 of cellisolation device 10 such that wells 122 overlie corresponding ones ofthe cell isolation regions 20. Structure 121 is then placed in directcontact with housing 11 to form a seal such that fluid communicationbetween cells 30 in adjacent cell isolation regions is inhibited. Asseen in FIG. 15A, the mated cell isolation device/cell expansion deviceis then inverted and cells 30 are transferred, for example bycentrifugal force, from cell isolation regions 20 to wells 122.

Wells 122 may be of any shape, but wells with circular or square shapedtop plan view (or transverse cross-sections) are preferred as theseshapes are commonly used in the industry. Nothwithstanding the shape,wells 122 of expansion device 120 preferably have a greater volume thancell isolation regions 20 such as, for example, having a greaterdiameter (as illustrated in FIG. 15A) or a greater depth (as illustratedin FIG. 15B). Preferably wells 122 have the same diameter as cellisolation regions 20 of cell delivery device 10. More preferably, thewells 122 of the expansion device 120 have a depth of about 2 mm. In oneembodiment, the lateral surfaces of well 122 are canted relative to oneanother or to the bottom surface of the well 122 in a test orientationof cell expansion device 120 as shown in FIG. 16. Canted wells 122permit easier access by allowing for lateral movement when performingdownstream experiments on cells 30 in wells 122 and may create a moreuniform interface with a small cell isolation region 20. On the otherhand, a well 122 that has lateral sides that are at a 90° angle to thebottom side of the well 122 in a test orientation of the cell expansiondevice 120 provide a greater volume, which may increase the timeavailable for the detection step to be described below.

Structure 121 is intended to define any number of wells 122, butpreferably defines the number of wells in a standard microtiter platesuch as a 24-, 96-, 384-, 768-, or 1536-well plate. More preferably,structure 121 defines the number of wells of a 385-well plate or1536-well plate. Approximately 2 μl of liquid can be held per well 122in a standard 1536 well plate. Larger well volumes can be achieved byextending the depth of the well 122 without compromising thecorrespondence between the pitch between adjacent wells 122 and thepitch between adjacent cell isolation regions 20.

In another embodiment of cell expansion device 120 as seen in FIG. 17,cell expansion device 120 a is configured such that the respective wells122 a are accessible from a bottom side 127 thereof in a testorientation of cell expansion device 120 a. Structure 123 a furtherdefines an entrance port 125 and optionally, an exit port 126. Thewells' 122 a accessibility from the bottom side 127 permits exchange ofmedia, thereby facilitating cell expansion and refeeding. Cells 30 areretained from passing through the access to the bottom side 127 of well122 a by a block such as a size constraint or semi-permeable membrane,such as a dialysis membrane, nitrocellulose or perforatedpolydimethylsialoxane, for example. The semi-permeable membrane of thisembodiment also allows for gas exchange while reducing the evaporationof liquid contained in each well 122 a. The initial flow-through of oldmedium may be drained by positive pressure, vacuum, or gravity tocapture liquid potentially containing antibody or another product of thedesired biological activity, on a surface designed forsampling/detection, e.g., nitrocellulose, glass, urethanes, rubber,molded plastic, or polydimethylsialoxane.

Preferably, the material used for the manufacture of cell expansiondevice 120 comprises any rigid or flexible material such as glass,urethanes, rubber, molded plastic, co-polymer or polymer, morepreferably urethanes, rubber, molded plastic, polymethylmethacrylate(PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™),polyvinylchloride (PVC), polydimethylsiloxane (PDMS), polysulfone, andthe like, and most preferably PDMS. Such materials are readilymanufactured from fabricated masters, using well known moldingtechniques, such as injection molding, embossing or stamping, or bypolymerizing the polymeric precursor material within the mold. Suchmaterials are preferred for their ease of manufacture, low cost anddisposability, as well as their general inertness to most extremereaction conditions.

Once the cells 30 are transferred from cell isolation device 10 to cellexpansion device 122, the cells 30 are incubated in the cell expansiondevice 122, for a sufficient amount of time to allow for proliferationof cells 30. Cell expansion device 122 is intended to be used at anybiologically viable temperature. Lowering the incubation temperature ofthe cells (i.e., from 37° C. to 18° C.) may, however, slow the metabolicprocesses of the cells 30 and reduce cell cloning time, thus extendingthe time for assay. Alternatively, media that is optimal for exhibitingthe desired biological activity that is to be screened but not optimalfor cell proliferation could also be used to extend the time for assay.

As illustrated in FIG. 16, after sufficient time has been allowed forcells 30 to grow and divide, a detection device 140 is utilized todetect the desired biological activity exhibited by the cells, such asantibody production. In the case of detecting antibody production, inone embodiment as illustrated in FIG. 16, detection device 140 generallycomprises a surface 141 defining an array of prongs 142 upon whichantigens are immobilized. In a preferred embodiment, surface 141 is asubstantially planar surface. In one embodiment, prongs 142 are coatedwith the specific antigen(s) used to immunize animals from which thecells being tested are derived. In use, detection device 140 is placedover structure 121 of cell expansion device 120 such that prongs 142 arein registration with wells 122. Prongs 142 are then immersed into wells122 of cell expansion device 120 in order to allow antigens immobilizedon prongs 142 to potentially bind to antibodies secreted/produced bycells, such as hybridoma cells in wells 120. Prongs 142 of detectiondevice 140 are then removed from well 122 of cell expansion device 120and then immersed into a solution of detectable secondary antibody.Secondary antibodies may be visualized by standard techniques known inthe art such as by enzymatic color reactions or fluorescence. A prongsurface may also be interfaced with mass spectrometer for additionalassay flexibility. Other methods of labeling and detecting theantibodies are well within the knowledge of one of skill in the art andtherefore are not discussed in detail herein.

The antibodies may be coated on prongs 142 through non-specificphysisorption or through the use of SAMs on prongs 142. For example, aparticular antigen may be specifically patterned on prong 142 by firstevaporating gold on a polyurethane prong, then adding a self-assemblingmonolayer onto which an antigen can be covalently linked. (See, e.g.,Mrksich et al., Annu. Rev. Biophys. Biomol. Struct., vol. 25 (1996)incorporated herein by reference). Additionally, other strategies forimmobilizing protein may be employed such as lysine linkage, cutinaselinkage, GST-ftision or GPI linkage.

In another embodiment of a detection device used to detect theproduction of antibodies from cells, such as, for example, hybridomacells, cell isolation device 10 additionally serves as a detectiondevice as shown in FIG. 18. In this embodiment, the bottoms of cellisolation regions 20 of cell isolation device 10 are coated with thespecific antigen(s) used to immunize animals from which the hybridomasbeing tested are derived. If the desired biological activity beingscreened is the production of a biological product, other thanantibodies, the bottoms of cell isolation regions 20 could be coatedwith a ligand that binds to the biological product (ligate) produced bythe desired biological activity that is being screened. Ligands include,for example, a peptide, hormone, enzyme, or carbohydrate. Cells 30 arethen delivered to cell isolation regions 20 and the cells 30 will beginto produce antibodies that will bind the antigens coated on cellisolation regions 20. Cells 30 are then transferred from the cellisolation regions 20 of cell isolation device 10 to the wells 122 ofcell expansion device 120. The antibodies bound to the antigens on thebottoms of cell isolation regions 20 are then detected using an ELISAsandwich or a variety of detection methods known in the art such assecondary antibody coupled to alkaline phosphatase, horse radishperoxidase, etc.

Although in the embodiment illustrated in FIG. 16, cell isolation region20 of the cell isolation device 10 actually becomes the detectiondevice, it is well within the scope of the present invention to utilizea separate detection device. In such an embodiment, the detection devicecomprises a structure defining a plurality of indentations sized toreceive cells from cell expansion device 120, wherein the bottom of theindentations are coated with a ligand that binds to the biologicalproduct (ligate) produced by the desired biological activity that isbeing screened. Such ligates include, for example, a protein, enzyme,peptide, carbohydrate or antibody. After cells are transferred from cellisolation device 10 to the cell expansion device 120, cells are, inturn, transferred to the indentations of the cell detection device wherethey will potentially express the biological product of the desiredbiological activity that is being screened and such biological productbinds its complementary binding pair member coated on the indentations.

Although the foregoing description of detection methods have beendescribed in reference to production of a biological product produced bythe desired biological activity being screened, the present inventioncontemplates the detection of any biological activity by any types ofcells. Such detection methods are well known to one of skill in the art.

It will be appreciated that the present disclosure is intended to setforth the exemplifications of the invention, and the exemplificationsset forth are not intended to limit the invention to the specificembodiments illustrated. The disclosure is intended to cover, by theappended claims, all such modifications as fall within the spirit andscope of the claims.

1. A method of screening cells for a desired biological activitycomprising: providing a cell isolation device defining a plurality ofcell isolation regions, each cell isolation region encompassing a recessand bioaffinity ligands, each of the cell isolation regions being sizedto isolate about one to about five cells on a surface within saidrecess, and the cell isolation regions positioned to have apredetermined pitch with respect to one another; delivering the aboutone cell to about five cells to each of the cell isolation regions;providing a cell expansion device defining a plurality of wells whereinthe position of the wells of the cell expansion device corresponds tothe predetermined pitch of the cell isolation regions; placing said cellisolation device in direct contact with said cell expansion device,inverting said cell isolation device, and transferring the about onecell to about five cells from said surface of said cell isolationregions to the wells; allowing the about one cell to about five cells toproliferate and exhibit a desired biological activity in the wells;assaying the desired biological activity of the one cell to about fivecells.
 2. The method of claim 1, wherein the step of deliveringcomprises delivering one cell to about five cells through a microfluidicchannel.
 3. The method of claim 2, wherein the microfluidic channel hasa diameter of about 100 microns.
 4. The method of claim 1, wherein therecess of each of the cell isolation regions defines a top conicalportion, and a bottom microwell portion adjoining the top conicalportion in a test orientation of the cell isolation device.
 5. Themethod of claim 1, wherein the recess of each of the cell isolationregions defines a top cylindrical portion, an intermediate conicalportion, and a bottom microwell portion, the intermediate conicalportion being disposed between and adjoining the top cylindrical portionand the bottom microwell portion.
 6. The method of claim 1, wherein thebioaffinity ligands are selected from the group consisting ofantibodies, self-assembled monolayers (SAMs), lectin, carbohydrate,transmembrane proteins, and antigens.
 7. The method of claim 1, whereinthe predetermine pitch of the cell isolation regions and thepredetermined pitch of the wells matches a pitch of a standardmicrotiter plate.
 8. The method of claim 1, wherein the cells aretransferred from the cell isolation device to the cell expansion deviceby centrifugal force.
 9. The method of claim 1, wherein the cells arehybridoma cells and the biological activity exhibited by the cells isantibody production.
 10. The method of claim 9, wherein assayingcomprises assaying the hybridoma cells for specific antibody productionby: providing a detection device comprising a member defining aplurality of prongs, the prongs being coated with specific antigens;immersing the prongs into the wells to allow potential binding betweenspecific antibodies produced by the hybridoma cells and the antigens;removing the prongs from the wells; immersing the prongs into adetection solution; detecting the presence of specific antibodies.
 11. Akit for screening cells for a desired biological activity, the kitcomprising: a cell isolation device defining a plurality of cellisolation regions, each cell isolation region encompassing a recess andbioaffinity ligands, each of the cell isolation regions being sized toisolate about one to about five cells therein, and the cell isolationregions further defining a predetermined pitch with respect to oneanother; and a cell expansion device defining a plurality of wellswherein the wells of the cell expansion device correspond to respectiveregions of the cell-isolation device, and the wells define apredetermined pitch matching the predetermined pitch of the cellisolation regions wherein when said cell isolation device is placed indirect contact with said cell expansion device and said cell isolationdevice is inverted said one cell to about five cells may be transferredfrom said surface of said cell isolation regions to said wells.
 12. Thekit of claim 11, further comprising a detection device for screening thecells for the desired biological activity.
 13. The kit of claim 11,wherein the recess of each of the cell isolation regions defines a topconical portion, and a bottom microwell portion adjoining the topconical portion in a test orientation of the cell isolation device. 14.The kit of claim 13, wherein the top conical portion has a depth ofabout 1 millimeter.
 15. The kit of claim 13, wherein the bottommicrowell portion has a diameter of about 10 microns to about 50 micronsand a depth of about 10 microns to about 50 microns.
 16. The kit ofclaim 15, wherein the bottom microwell portion has a diameter of about20 microns and a depth of about 20 microns.
 17. The kit of claim 11,wherein the recess of each of the cell isolation regions defines a topcylindrical portion, an intermediate conical portion, and a bottommicrowell portion, the intermediate conical portion being disposedbetween and adjoining the top cylindrical portion and the bottommicrowell portion.
 18. The kit of claim 17, wherein the cylindricalportion has a diameter of about 2 millimeters and a depth of about 3millimeters.
 19. The kit of claim 17, wherein the conical portion has adepth of about 1 millimeters.
 20. The kit of claim 17, wherein thebottom microwell portion has a diameter of about 10 microns to about 50microns and a depth of about 10 microns to about 50 microns.
 21. The kitof claim 11, wherein the pitch of the cell isolation regions and thepitch of the wells match a pitch of a standard microtiter plate.